Most kidney stones form as aggregates of calcium oxalate monohydrate (COM) crystals under the influence of urinary macromolecules, but currently, no urine test can prospectively identify stone formers. Prior work in our laboratory has shown that the urinary macromolecular mixtures from stone formers have low net negative charge, compared to normal healthy adults. Also, it has been shown that macromolecular aggregation occurs when the net charge is near zero; either by mixing equal protions of polycations and polyanions or by reducing the number of negative charges per chain on polyanions, such as Tamm-Horsfall Protein, and that macromolecular aggregate formation causes COM crystal aggregation. This proposal tests the hypothesis that stone formers can be identified by the presence of low net negative charge in their urinary macromolecules, and that this condition is linked to macromolecular aggregation, crystal aggregation, and stone formation. This proposal will test this hypothesis by following a research protocol with 2 Specific Aims: (1) Verification of low net negative charge in urinary macromolecules as the disease marker for stone formers, and correlation with phenotype. Urinary macromolecules from recurrent COM stone formers and normal controls will be isolated and characterized for their net negative charge by Colloidal Titration to verify that low net negative charge is a marker for stone disease, and confirm a simplified sample handling procedure recently developed in our laboratory. These same samples will also be fully characterized with respect to their effect on COM crystal nucleation, aggregation, and growth to define phenotypic cohorts; subsets of patients that are likely to share a common defect in their urinary macromolecules to facilitate identification under Specific Aim 2. Familial cohorts will also be studied for this purpose, since they are likely characterized by a single defect. (2) Identification of the macromolecular structural defect(s) associated with low net negative charge and stone formation. Specific macromolecular components (or combinations thereof) responsible for specific functional defects will be identified in stone formers using various techniques of proteomic analyses, specifically 2 dimensional gel electrophoresis methods and MALDI-TOF mass spectrometry. These methods will identify whether the low net negative charge observed in the urinary macromolecular mixtures of stone formers derives from 1) the presence of additonal polycationic macromolecules, 2) the absence of critical polyanionic macromolecules, or 3) reduction of net charge on critical polyanion macromolecules compared to those in the urine from normal healthy adults, and therefore identify the cause(s) of disease. We anticipate that more than one type of defect will be found, since many different macromolecules are associated with kidney stones. PUBLIC HEALTH RELEVANCE: The successful completion of this proposal will yield a diagnostic method, specifically Colloidal Titration, for prospectively identifying stone formers, and allowing for dietary and lifestyle counselling to prevent or delay onset of disease. Identification of altered protein structures leading to the changes observed in the Colloidal Titration assay will help to confirm our model of the stone forming processes, providing the basis for the intelligent design of new therapeutic agents that truly prevent disease recurrence, rather than reduce it, as in current treatments.